Ancient Plasmodium genomes shed light on the history of human malaria.

Malaria-causing protozoa of the genus Plasmodium have exerted one of the strongest selective pressures on the human genome, and resistance alleles provide biomolecular footprints that outline the historical reach of these species1. Nevertheless, debate persists over when and how malaria parasites emerged as human pathogens and spread around the globe1,2. To address these questions, we generated high-coverage ancient mitochondrial and nuclear genome-wide data from P. falciparum, P. vivax and P. malariae from 16 countries spanning around 5,500 years of human history. We identified P. vivax and P. falciparum across geographically disparate regions of Eurasia from as early as the fourth and first millennia BCE, respectively; for P. vivax, this evidence pre-dates textual references by several millennia3. Genomic analysis supports distinct disease histories for P. falciparum and P. vivax in the Americas: similarities between now-eliminated European and peri-contact South American strains indicate that European colonizers were the source of American P. vivax, whereas the trans-Atlantic slave trade probably introduced P. falciparum into the Americas. Our data underscore the role of cross-cultural contacts in the dissemination of malaria, laying the biomolecular foundation for future palaeo-epidemiological research into the impact of Plasmodium parasites on human history. Finally, our unexpected discovery of P. falciparum in the high-altitude Himalayas provides a rare case study in which individual mobility can be inferred from infection status, adding to our knowledge of cross-cultural connectivity in the region nearly three millennia ago.

DNA polymerase kappa from Trypanosoma cruzi localizes to the mitochondria, bypasses 8-oxoguanine lesions and performs DNA synthesis in a recombination intermediate.

DNA polymerase kappa (Pol kappa) is a low-fidelity polymerase that has the ability to bypass several types of lesions. The biological role of this enzyme, a member of the DinB subfamily of Y-family DNA polymerases, has remained elusive. In this report, we studied one of the two copies of Pol kappa from the protozoan Trypanosoma cruzi (TcPol kappa). The role of this TcPol kappa copy was investigated by analysing its subcellular localization, its activities in vitro, and performing experiments with parasites that overexpress this polymerase. The TcPOLK sequence has the N-terminal extension which is present only in eukaryotic DinB members, but its C-terminal region is more similar to prokaryotic and archaeal counterparts since it lacks C(2)HC motifs and PCNA interaction domain. Our results indicate that in contrast to its previously described orthologues, this polymerase is localized to mitochondria. The overexpression of TcPOLK increases T. cruzi resistance to hydrogen peroxide, and in vitro polymerization assays revealed that TcPol kappa efficiently bypasses 8-oxoguanine lesions. Remarkably, our results also demonstrate that the DinB subfamily of polymerases can participate in homologous recombination, based on our findings that TcPol kappa increases T. cruzi resistance to high doses of gamma irradiation and zeocin and can catalyse DNA synthesis within recombination intermediates.

Isolation and characterization of HC1: a novel human DNA repair gene.

Nucleotide excision repair (NER) acts on a broad spectrum of large lesions, while base excision repair removes individual modified bases. Although both processes have been well studied in human cells, novel genes involved in these DNA repair pathways have been described. Using a heterologous complementation approach, we identified a fetal human cDNA that complemented two Escherichia coli mutants that are defective in 3-methyl adenine glycosylase and in three endonucleases, all of which are enzymes with important roles in base excision repair. The central cDNA open reading frame complemented NER mutant strains and promoted an increase in survival rate of bacteria exposed to UV light. The corresponding protein was able to restore nucleotide-excision-repair activity when added to a cell extract from Chinese hamster ovary cells deficient in the ERCC1 protein, an enzyme known to promote incision at the 5' end of the lesion during NER. In contrast, that protein was not able to complement XPG Chinese hamster ovary cells deficient in the 3' incision step of NER. These data indicate a new human repair gene, which we named HC1; it is involved in the recognition of two kinds of DNA lesions and it contributes to the 5' DNA incision step in NER.

Schistosoma mansoni: Microarray analysis of gene expression induced by host sex.

Schistosoma mansoni is a digenetic trematode and a human parasite responsible for high social and economic impact. Although some authors have studied the effect of host hormones on parasites, not much is known about the effects of host sex on gene expression in Schistosomes. In order to study gene transcripts associated with the host sex, we compared the gene expression profiles of both male and female unisexual adult S. mansoni parasites raised on either male or female hosts, using DNA microarrays. Our results show that host sex caused differential expression of at least 11 genes in female parasites and of 134 in male parasites. Of the differentially expressed genes in female worms, 10 were preferentially expressed in female worms from male mice, while of the 134 differentially expressed genes in male parasites, 79 (59%) were preferentially expressed in worms from female mice. Further investigation of the role of each of those genes will help understand better their importance in the pathogenesis of Schistosomiasis.

BNDb--Biomolecules Nucleus Database: an integrated proteomics and transcriptomics database.

Proteomics correspond to the identification and quantitative analysis of proteins expressed in different conditions or life stages of a cell or organism. Methods used in proteomics analysis include mainly chromatography, two-dimensional electrophoresis and mass spectrometry. Data generated in proteomics analysis vary significantly, and to identify a protein it is often necessary to perform a series of experiments, comparing its results to those found in proteomics databases. Existing proteomics databases are usually related to only one type of experiment or represent processed results, not raw data. Therefore, proteomics researchers frequently have to resort to several data repositories in order to be able to perform the identification. In this paper, we propose an integrated proteomics and transcriptomics database that stores raw and processed data, which are indexed allowing them to be retrieved together or individually. The proposed database, dubbed BNDb for Biomolecules Nucleus Database, is implemented using an MySQL server and is being used to store data from the parasite Schistosoma mansoni, the scorpion Tittyus serrulatus and the spider Phoneutria nigriventer. The database construction uses a relational approach and data indexes. The data model proposed uses groups of tables for each data subtype, which store details regarding the experimental procedure as well as raw data, analysis results and associated publications. BNDb also stores transcriptomics data publicly available which are associated with identifications performed on new samples. By using BNDb, we expect not only to contribute to proteomics research but also to provide a useful service for the scientific community.

Analysis of DNA polymerase activity in vitro using non-radioactive primer extension assay in an automated DNA sequencer.

Although different DNA polymerases have distinct functions and substrate affinities, their general mechanism of action is similar. Thus, they can all be studied using the same technical principle, the primer extension assay employing radioactive tags. Even though fluorescence has been used routinely for many years for DNA sequencing, it has not been used in the in vitro primer extension assay. The use of fluorescence labels has obvious advantages over radioactivity, including safety, speed and ease of manipulation. In the present study, we demonstrated the potential of non-radioactive in vitro primer extension for DNA polymerase studies. By using an M13 tag in the substrate, we can use the same fluorescent M13 primer to study different substrate sequences. This technique allows quantification of the DNA polymerase activity of the Klenow fragment using different templates and under different conditions with similar sensitivity to the radioactive assay.

Evaluation of cDNA libraries from different developmental stages of Schistosoma mansoni for production of expressed sequence tags (ESTs).

A comparative study of the gene expression profile in different developmental stages of Schistosoma mansoni has been initiated based on the expressed sequence tag (EST) approach. A total of 1401 ESTs were generated from seven different cDNA libraries constructed from four distinct stages of the parasite life cycle. The libraries were first evaluated for their quality for a large-scale cDNA sequencing program. Most of them were shown to have less than 20% useless clones and more than 50% new genes. The redundancy of each library was also analyzed, showing that one adult worm cDNA library was composed of a small number of highly frequent genes. When comparing ESTs from distinct libraries, we could detect that most genes were present only in a single library, but others were expressed in more than one developmental stage and may represent housekeeping genes in the parasite. When considering only once the genes present in more than one library, a total of 466 unique genes were obtained, corresponding to 427 new S. mansoni genes. From the total of unique genes, 20.2% were identified based on homology with genes from other organisms, 8.3% matched S. mansoni characterized genes and 71.5% represent unknown genes.

Characterization of a Schistosoma mansoni gene encoding a homologue of the Y-box binding protein.

We have cloned and characterized a Schistosoma mansoni cDNA encoding a basic protein homologous to the human Y-box binding protein 1 (YB-1). The 1.3-kb S. mansoni YB-1 transcript, which was shown to be expressed in various stages of the parasite life cycle, codes for a protein of 217 amino acids containing, towards its N-terminus, a nucleic acid binding motif, known as the cold-shock domain (CSD). This domain is 64% identical to the cold-shock domain of other members of the Y-box binding protein family and 43% identical to the cold-shock protein CspA of Escherichia coli. In S. mansoni YB-1, the cold-shock domain possess some structural characteristics that permit dimer formation as occurs in the Bacillus subtilis cold-shock protein CspB. The C-terminal region of S. mansoni YB-1 differs from the other Y-box binding proteins because of the presence of tandem repeats of Arg and Gly, suggesting the formation of a fibroin-like beta-sandwich structure. This novel folding pattern for the C-terminus of S. mansoni YB-1 might suggest a distinct specific function for this protein in the parasite.

Identification of new Schistosoma mansoni genes by the EST strategy using a directional cDNA library.

A directional size-selected cDNA library constructed from Schistosoma mansoni (Sm) adult worm RNA was used for the generation of expressed sequence tags (EST). From one or both ends of 429 distinct cDNA clones 607 EST were obtained. Of these, only 16% were previously known Sm genes. More than 22% of the clones had matches with entries for other organisms in the databases. These new Sm genes constituted a broad range of transcripts distributed among cytoplasmic structural and regulatory proteins, enzymes, membrane, nuclear and secretory proteins, and proteins with other functions. Almost 33% of the clones had no significant database matches and thus potentially represent Sm-specific genes. Among the latter, several clones, as judged by their redundancy in the library, appear to represent abundant transcripts. The data, taken as a whole, more than double the number of Sm genes identified by nucleotide sequencing and indicate the potential value of the adoption of genome sequencing strategies for the rapid increase in knowledge of complex disease-causing organisms.

Yeast artificial chromosome (YAC)-based genome mapping of Schistosoma mansoni.

Schistosoma mansoni has 7 pairs of autosomal chromosomes and one pair of sex chromosomes (ZZ for a male worm and ZW for a female), of a haploid genome size of 2.7 x 10(8) bp. We initiated the molecular genetic approach for the detailed characterization and understanding of the evolutionary biology of schistosomes. We have constructed a yeast artificial chromosome (YAC)-library with partially digested parasite genomic DNA, and the chromosome location of each insert was detected by fluorescent in situ hybridization (FISH). The library contains > 2283 clones with an average insert size of 358 kb, which represents a 2.6-fold coverage of the genome (> 7.2 x 10(8) bp). 100 randomly selected YAC clones were localized by FISH and found to be distributed widely among all chromosomes. The assembly of 14 YACs distributed almost the whole region of chromosome 3. Generated expressed sequenced tags (ESTs) derived from a unidirectional cDNA library were also used for further characterization of the YAC inserts. These results indicate that an extensive contig assembly of the entire chromosomes and a reasonably detailed gene map should be feasible in the near future.

Y-box binding protein from Schistosoma mansoni: interaction with DNA and RNA.

A Schistosoma mansoni homologue of the human Y-box binding protein (SMYB1), as well as truncated proteins containing its N-terminal Cold Shock Domain (CSD) or its C-terminal domain (TAIL) were cloned into the p-MAL-c2 expression vector and produced in Escherichia coli. In order to characterize the interactions of these proteins to an inverted CCAAT motif present in a number of gene promoters, their binding to DNA was measured by Electrophoretic Mobility Shift Assays. SMYB1 bound to single- and double-stranded DNA containing the CCAAT motif and could bind also to RNA. The truncated CSD and TAIL domain proteins bound to dsDNA and RNA, but exhibited distinct binding patterns. Protein-DNA interaction was also investigated in vivo, using the Yeast One-Hybrid System. The plasmid constructs were GSTTRI, a DNA fragment composed of three copies of the CCAAT motif of the S. mansoni glutathione S-transferase gene promoter and four oligonucleotides spanning different regions of the S. mansoni p14 gene promoter. None of the yeast clones transformed with the above plasmids was able to grow in selective medium or to activate the transcription of the HIS3 reporter gene, suggesting that SMYB1 could not interact with these promoters in vivo.

Analysis of the gene expression profile of Schistosoma mansoni cercariae using the expressed sequence tag approach.

ESTs constitute rapid and informative tools with which to study gene-expression profiles of the diverse stages of the schistosome life cycle. Following a comprehensive EST study of adult worms, analysis has now targeted the cercaria, the parasite larval form responsible for infection of the vertebrate host. Two Schistosoma mansoni cercarial cDNA libraries were examined and partial sequence obtained from 957 randomly selected clones. On the basis of database searches, 551 (57.6%) ESTs generated had no homologs in the public databases whilst 308 (32.2%) were putatively identified, totaling 859 informative ESTs. The remaining 98 (10.2%) were uninformative ESTs (ribosomal RNA and non-coding mitochondrial sequences). By clustering analysis we have identified 453 different genes. The most common sequences in both libraries represented Sm8 calcium binding protein (8% of ESTs), fructose-1,6-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, cytochrome oxidase subunit 1, ATP guanidine kinase and triose phosphate isomerase. One hundred and nineteen identified genes were sorted into 11 functional categories, with genes associated with energy metabolism being the most abundant (13%) and diverse. The diversity and abundance of genes associated with the transcription/translation machinery and with regulatory/signaling functions were also marked. A paramyosin transcript was identified, indicating that this gene is not exclusively expressed in adult worms and sporocysts (as had been suggested previously). The possible physiological relevance to cercariae of the presence of transcripts with homology to calcium binding proteins of the EF-hand superfamily, Gq-coupled rhodopsin photoreceptor, rod phosphodiesterase 8 subunit and peripheral-type benzodiazepine receptor is discussed.

The Schistosoma gene discovery program: state of the art.

Schistosoma are dioecious digenetic trematodes carrying a large (270 Mb) genome. Gaining knowledge about the genome of these parasites is of importance for the understanding of their biology, mechanisms of drug resistance and antigenic variation that determine escape from the host's immune system. This review will provide an update on the Schistosoma Gene Discovery Program, which is part of the Schistosoma Genome Project created in 1992. One of the main objectives of this program is the discovery and characterisation of new genes of Schistosoma mansoni and Schistosoma japonicum in an attempt to search for new targets for drugs and vaccine development. The success of the Schistosoma Gene Discovery Program is demonstrated by the number of catalogued genes, that now reaches 15 to 20% of the full gene complement of its genome.

Nucleic acid binding properties of SmZF1, a zinc finger protein of Schistosoma mansoni.

During its life cycle, the flat worm Schistosoma mansoni is exposed to diverse environmental conditions and changes its morphological form. Each change calls for distinct patterns of gene expression. In order to understand the regulation of gene expression, it is necessary to identify regulatory elements in the promoter region of genes, and DNA transacting factors that control transcription. Zinc finger protein domains are responsible for transcription regulation of diverse genes in a wide range of organisms and are also involved in the promotion of protein-protein interactions. A transcript homologous to zinc finger gene sequences was isolated from a S. mansoni adult worm cDNA library and named SmZF1. It codes for a protein of 164 amino acids presenting three C(2)H(2) type zinc finger motifs. The recombinant SmZF1 protein was expressed and used on electrophoretic mobility shift assays to investigate the binding specificity of SmZF1 for DNA and RNA oligonucleotides. Our results demonstrated that SmZF1 binds both ds and ss DNA oligonucleotides, with an apparent preference for the specific D1-3DNA oligonucleotide, and also binds RNA oligonucleotides with lower affinity. Although we found that SmZF1 recognises DNA and RNA oligonucleotides not containing putative target sites, SmZF1 binds preferentially to sequence specific sites. Furthermore, unrelated oligonucleotides are not able to abolish this interaction. In silico studies identified putative SmZF1 binding sites in the complete genome of three model organisms and in partial genome sequences of S. mansoni. Six Drosophila genes presented these binding sites in their promoter region, indicating that they might be controlled by transcription factors containing zinc fingers motifs. Taken together, these results suggest that SmZF1 acts as a putative transcription factor of S. mansoni.

Biological activities of a human amniotic membrane interferon.

In order to characterize further the human amniotic membrane interferon (IFN-AM), an interferon antigenically unrelated to human IFN-alpha, -beta, and -gamma or TNF, we analysed its biological activities. Here, we present direct evidence of its ability to affect cell growth and to induce the IFN-stimulated genes (ISGs) 6-16 and 2'-5' oligoadenylate synthetase (OAS), in addition to its crossed anti-viral activity. The cellular growth arrest effect of IFN-AM was dose-dependent and paralleled that of IFN-beta. IFN-AM was also able to inhibit thymidine incorporation into DNA, similar to IFN-beta. The mRNA induction of 6-16 gene with IFN-AM treatment reached its highest level at 500 IU/ml and remained constant up to 2000 IU/ml. Conversely, 2'-5' OAS mRNA induction was dose-dependent, with the maximum level detected at 2000 IU/ml of IFN-AM treatment. The time course of mRNA accumulation by ISGs with IFN-AM (500 IU/ml) stimulation was also investigated. Gene induction reached a maximum at 16 h after IFN treatment for 2'-5' OAS and at 48 h for the 6-16 gene. IFN-AM and human IFN-alpha induced similar levels of the OAS enzyme. IFN-AM also showed small but significant activity in bovine cells. In conclusion, the amniotic membrane IFN here studied showed both anti-cellular activity and the ability to stimulate ISG-transcriptional activation in a similar manner to IFN-beta. In addition, IFN-AM was also as able to induce the expression of the enzyme 2'-5' OAS, as did IFN-alpha. Lastly, amniotic IFN showed a significant cross-species anti-viral activity, which was different from both human IFN-alpha and -beta. Taken together, these data strongly suggest that IFN-AM is a novel sub-type I IFN.

Characterization of a Schistosoma mansoni homologue of the gene encoding the breast basic conserved protein 1/L13 ribosomal protein.

The Schistosoma mansoni gene sequence encoding the breast basic conserved protein 1/ribosomal protein L13 has been isolated from an adult worm cDNA library using the Expressed Sequence Tag strategy. The cDNA codes for a putative protein of 184 amino acids which is approximately 55% identical to other eukaryotic L13 ribosomal proteins. A PCR amplified genomic fragment containing the coding region of the gene was seen to possess only a single large intron interrupting the open reading frame. Studies of gene expression by RT-PCR showed the transcript is expressed in distinct stages of the parasite life cycle. The cDNA was also hybridized with an ordered cosmid library of S. mansoni and the identified cosmids were mapped to chromosomes 3 and W by chromosomal in situ suppression hybridization.

Identification of genes encoding Schistosoma mansoni antigens using an antigenic sequence tag strategy.

Another approach for the identification of genes that code for antigenic products is described using an antigenic sequence tag (AST) strategy. A Schistosoma mansoni adult worm cDNA library was screened with affinity chromatography-purified immunoglobulins from infected human sera and a mild oxidation treatment with sodium periodate. From 1 or both ends of 30 cDNA clones, 30 ASTs were obtained. Of these, 22 were previously known Sm antigens. One clone had matches with entries for other organisms in the databases and 6 had homology with Sm-expressed sequence tags (EST) entries. These clones, together with another 1 that had no significant database matches, were considered new antigenic genes in S. mansoni. The strategy proved to be efficient for the identification of genes that could be used for immunological studies and evaluation as vaccine candidates.

Draft Genome Sequence of the Probiotic Yeast Saccharomyces cerevisiae var. boulardii Strain ATCC MYA-796.

Saccharomyces boulardii is the only yeast approved as a probiotic for human consumption. Here, we report the draft genome sequence of the strain ATCC MYA-796, derived from the French Ultra Levure probiotic drug. The genome has a size of 11.6 Mb with 5,305 putative open reading frames predicted.

Effects of chronic passive smoking on the regeneration of rat femoral defects filled with hydroxyapatite and stimulated by laser therapy.

Defects associated with bone mass loss are frequently treated by autogenous bone grafting. However, synthetic biomaterials such as calcium phosphate ceramics can substitute autologous grafts as long as they are biocompatible with bone tissue. In addition, low-level laser therapy (LLLT) is used to enhance bone regeneration by stimulating the local microcirculation and increasing the synthesis of collagen by bone cells. However, bone health is fundamental for osseointegration of the graft and bone repair. In this respect, excessive tobacco consumption can compromise expected outcomes because of its deleterious effects on bone metabolism that predispose to the development of osteoporosis. The objective of this study was to evaluate the regeneration of bone defects implanted with biomaterial and stimulated by LLLT in rats submitted to passive cigarette smoking. Porous hydroxyapatite granules were implanted into critical-size defects induced experimentally in the distal epiphysis of the right femur of 20 female Wistar rats submitted to passive smoking for 8 months in a smoking box. The defect site was irradiated with a gallium-arsenide laser at an intensity of 5.0 J/cm2. The animals were divided into four groups: control (non-smoking) rates submitted (G2) or not (G1) to laser irradiation, and smoking rats submitted (G4) or not (G3) to laser irradiation. The animals were sacrificed 8 weeks after biomaterial implantation. The right femurs were removed for photodocumentation, radiographed, and processed for routine histology. The results showed good radiopacity of the implant site and of the hydroxyapatite granules. Histologically, formation of new trabecular bone was observed adjacent to the hydroxyapatite granules in G1 and G2. In G3 and G4, the granules were surrounded mainly by connective tissue. In conclusion, passive smoking compromised bone neoformation in the defects and the LLLT protocol was not adequate to stimulate local osteogenesis.

Sequencing and identification of expressed Schistosoma mansoni genes by random selection of cDNA clones from a directional library.

We have initiated a gene discovery program in Schistosoma mansoni based on the technique of Expressed Sequence Tags (ESTs), i.e. partial sequences of cDNAs obtained from single passes in automatic DNA sequences. ESTs can be used to identify genes on the basis of their homology with sequences from other species deposited in DNA or protein databases. Transcripts with sequences without matches in the databases may represent novel parasite-specific genes. This approach has shown to be very efficient and in less than two years a broad range of novel genes has already been ascertained, more than doubling the number of known S. mansoni genes.